The goal of this project is the identification of nonlethal mutations in th DEN virus genome which when incorporated into the infectious cDNA clone (project #Z01 BK 07001-01) will reduce virulence. Four related studies are in progress or concluding: (1) Nuclear localization of the dengue virus capsid protein. The DEN4 capsid (C) was expressed in LLCMK2 cells using a vaccinia virus system. We identified a nuclear localization signal (NLS) i the C sequence within a domain which contains a predicted consensus sequenc for an NLS. C appeared to contain at least one additional NLS. Current experiments are directed at its identification. These data suggest nucleus- dependent steps in the replication of flaviviruses. (2) Ubiquitination of the dengue virus capsid protein. Conjugation of multiple ubiquitin molecules to a Lys residue is thought to target a protein for degradation a a rate related to the identity of the N-terminal amino acid. (The "N-end" rule.) C was multiply ubiquitinated during in vitro synthesis. C containing destabilizing N-end residues encoded by mutated cDNAs were not unstable, a surprising finding. The association of C with RNA, may prevent degradation. (3) The mode of association of the flavivirus structural glycoproteins. Association of prM and E on nascent virus is essential for their transport from the ER to the trans Golgi. The role of conserved portions of the ectodomains and of the hydrophobic transmembrane domains in prM and E in the formation of heterodimers and exocytic transport is being evaluated in vivo and in vitro. (4) Effect of mutations in the DEN NS2B protein on protease activity of NS3. Most cleavages of the nonstructural protein (NS) region of the DEN virus polyprotein are effected by a virus- encoded protease composed of NS2B+NS3. NS3 contains the protease active site; the role of NS2B is not established. To determine the requirement of NS2B for the protease activity of NS3, we mutagenized a 40-amino-acid domai in NS2B indispensable for protease activity. 60 mutants have been isolated and recombined for the expression in concert with NS3. Autocleavage at the NS2B-NS3 site is being analyzed in vivo and in vitro.